Conformational Rearrangements in the Redox Cycling of NADPH-Cytochrome P450 Reductase from Sorghum bicolor Explored with FRET and Pressure-Perturbation Spectroscopy

Abstract

NADPH-cytochrome P450 reductase (CPR) from Sorghum bicolor (SbCPR) serves as an electron donor for cytochrome P450 essential for monolignol and lignin production in this biofuel crop. The CPR enzymes undergo an ample conformational transition between the closed and open states in their functioning. This transition is triggered by electron transfer between the FAD and FMN and provides access of the partner protein to the electron-donating FMN domain. To characterize the electron transfer mechanisms in the monolignol biosynthetic pathway better, we explore the conformational transitions in SbCPR with rapid scanning stop-flow and pressure-perturbation spectroscopy. We used FRET between a pair of donor and acceptor probes incorporated into the FAD and FMN domains of SbCPR, respectively, to characterize the equilibrium between the open and closed states and explore its modulation in connection with the redox state of the enzyme. We demonstrate that, although the closed conformation always predominates in the conformational landscape, the population of open state increases by order of magnitude upon the formation of the disemiquinone state. Our results are consistent with several open conformation sub-states differing in the volume change (ΔV⁰) of the opening transition. While the ΔV⁰ characteristic of the oxidized enzyme is as large as -88 mL/mol, the interaction of the enzyme with the nucleotide cofactor and the formation of the double-semiquinone state of CPR decrease this value to -34 and -18 mL/mol, respectively. This observation suggests that the interdomain electron transfer in CPR increases protein hydration, while promoting more open conformation. In addition to elucidating the functional choreography of plant CPRs, our study demonstrates the high exploratory potential of a combination of the pressure-perturbation approach with the FRET-based monitoring of protein conformational transitions.

Keywords: FRET; Sorghum bicolor; conformational change; cytochrome P450 reductase; pressure-perturbation spectroscopy; protein hydration; reduction kinetics; stop-flow spectroscopy.

Figure A1

Spectra of absorbance (dashed lines) and fluorescence (solid lines) of DY520-XL (red) and DY731 (blue). The absorbance spectrum of SbCPR is shown in a black dashed line.

Figure A2

Spectra of fluorescence of SbCPR-2DY (red) and an equimolar mixture of single labeled SbCPR-DY520XL with SbCPR double-labeled with MBBR and DY-731 (black) taken at 5 °C with excitation at 505 nm. The spectra shown in the main panel are normalized to the same integral intensity of fluorescence. The inset shows the donor emission bands unscaled.

Scheme 1

Scheme of electron transfer events in CPR.

Figure 1

Structural model of SbCPR based on a combination of the resolved X-ray structure of the FAD domain (PDB ID: 7SUX) with the model of FMN domain and the connecting loop built with AlphaFold. The two modification-accessible cysteine residues are shown as yellow spheres. FMN and FAD domains are pink and light blue, respectively, and the connecting loop is green. FAD and FMN molecules are shown as orange stick models. The N-terminal transmembrane helix of the enzyme is not shown.

Figure 2

Changes in the spectra of absorbance of SbCPR in the process of its NADPH-dependent reduction. Panel (a) exemplifies a series of absorbance spectra recorded in a rapid-scanning stop-flow experiment. Panel (b) shows the spectra (eigenvectors) of the first (red) and second (blue) Principal Components obtained with PCA of the above dataset, and panel (c) represents the respective sets of eigenvalues plotted against time in semi-logarithmic coordinates. Solid lines in this panel correspond to the approximations of the kinetic curves with the three-exponential equation.

Figure 3

Changes in absorbance of SbCPR at 380, 454, and 595 nm in the process of its NADPH-dependent reduction. Panel (a) shows the initial parts of the curves in linear coordinates. In panel (b), the same datasets are plotted versus the logarithm of time in the entire time range of the experiment. Solid lines correspond to the approximations of the kinetic curves with the three-exponential equation.

Figure 4

Changes in the fluorescence of SbCPR-2DY in the process of its NADPH-dependent reduction studied by rapid-scanning stop-flow spectroscopy. Panel (a) shows a representative set of spectra of fluorescence with excitation at 505 nm registered at different time points. Here, the gray arrows indicate the direction of the changes during the initial 100 ms of the process. Black arrows indicate the direction of the changes during the latter phase of reduction. A series of spectra of fluorescence recorded at the same time points with the direct excitation of the acceptor at 617 nm is shown in panel (b). Panel (c) illustrates the spectral changes during the reduction as a 3D plot of fluorescence spectra (excitation at 505 nm) normalized on the integral fluorescence intensity. A kinetic curve of the changes in FRET efficiency during the reduction is shown in panel (d) in semi-logarithmic coordinates. The solid line shown in this plot corresponds to the approximation of this data set by a three-exponential equation.

Figure 5

Pressure-induced changes in the spectra of fluorescence of SbCPR-2DY. Panels (a,b) show the series of spectra recorded at increasing pressure with the oxidized (a) and reduced (b) enzyme. The spectra shown in solid lines were recorded at 1, 300, 600, 900, 1200, 1500, 1800, 2100, and 2400 bar. In the case of the reduced enzyme, this set is complemented with the spectra recorded at 3000, 3600, and 4200 bar. The spectra shown in thick gray dashed lines were recorded after decompression to the ambient pressure. The pressure dependencies of FRET efficiency obtained with four different enzyme states are exemplified in the main plot of the panel (c). Here, the solid lines show the approximations of the datasets with Equation (6). The inset in this panel exemplifies the pressure dependencies of the relative fluorescence intensity of the double-labeled enzyme with excitation at 617 nm (filled circles, solid line) and single-labeled SbCPR-DY520XL with excitation at 505 nm (open circles, dashed line). Lines show the approximations of the datasets with Equation (6).